The long-term goal of my research is to study basic mechanisms of synaptic formation and elimination. In this proposal, the regeneration and development of neuromuscular junctions will be studied with an innovative approach, combining intracellular recordings and freeze-fracture. The main objectives are to study (1) the formation of the active zone, which is thought to be the site of neurotransmitter release, and (2) the intramembrane stuctural changes associated with the formation and the elimination of polyneuronal innervation. The research will be divided into two projects: (I) The reinnervation of neuromuscular junctions in the frog. I have already studied denervation of these junctions. Muscles following reinnervation will be studied with intracellular recordings. Then, nerves will be stimulated in fixative and the same muscles will be freeze-fractured. I will study (a) how active zones re-form into the normal unique oranization at regions just opposite junctional folds, (b) how the organization of regenerating active zones is related to the exocytosis of synaptic vesicles, and (c) the intramembrane structure in polyneuronally innervated junctions, which remain stable indefinitely. (II) The development of neuromuscular junctions in the tadpole. I will use similar methods and study similar aspects of the formation of junctions to those described in (I). Since, during development in the tadpole, junctional holds have just begun to form and polyneuronal innervation is eliminated, the comparative study between (I) and (II) will give insights into the relation between active zones and junctional folds, and into the mechanism of th elimination of redundant terminals. The proposed research will provide a more fundamental understanding of the nature of the active zone and the process of transmitter release. It will also provide molecular pictures of the maturation, suppression and possibly even recognition of synapses. Understanding of the basic mechanisms of synaptic formation and elimination will undoubtedly have a strong impact on learning the underlying mechanism of development of intelligence and certain developmental neurological disorders.